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| United States Patent |
5,506,134
|
|
Soule
, et al.
|
April 9, 1996
|
Hypridoma and monoclonal antibody which inhibits blood coagulation
tissue factor/factor VIIa complex
Abstract
Coagulation protein antagonists are disclosed, which include
monoclonal-type antibodies and related cell lines disclosed for the
production of specific, neutralizing antibodies against factors VII and
VIIa and the tissue factor/factor VIIa bimolecular complex, which
antibodies are useful for the prevention or treatment of thrombotic and
related diseases, for immunoaffinity isolation and purification of factors
VII and VIIa and the tissue factor/factor VIIa complex, and for
determination of factors VII or VIIa and the tissue factor/factors VII or
VIIa complex in a biological sample.
| Inventors:
|
Soule; Howard R. (Encinitas, CA);
Brunck; Terence K. (San Diego, CA)
|
| Assignee:
|
Corvas International, Inc. (San Diego, CA)
|
| Appl. No.:
|
759443 |
| Filed:
|
September 13, 1991 |
| Current U.S. Class: |
435/337; 530/387.1; 530/388.1; 530/388.25 |
| Intern'l Class: |
C12N 005/12; C07K 016/00; C07K 016/18 |
| Field of Search: |
530/388.25,387.1,388.1,388.25
435/240.27,70.21
|
References Cited
U.S. Patent Documents
| 4382083 | May., 1983 | Thomas | 424/101.
|
| Foreign Patent Documents |
| 0136835 | Sep., 1984 | EP.
| |
| WO91/07432 | May., 1991 | EP.
| |
| 8912469 | Dec., 1989 | WO.
| |
Other References
Carson et al., Blood, 70:490-493, Aug. 1987.
Takase, et al., J. Clin Pathol, 41:337-341, 1988.
Warr et al., 75 Blood 1481, 1990.
Carson et al., 66 Blood 152, 1985.
Drake et al., 134 American Journal of Pathology 1807, 1989.
Drake et al., 109 Journal of Cell Biology 389, 1989.
"Preliminary Characterization of a Panel of Monoclonal Antibodies to Human
Factor VII", Coppola et al., Curr. Stud. Hematol. Blood Transfus. (1991)
58:187-193.
"Monoclonal Antibody (VII-M31) to Bovine Factor VII: A Specific Epitope in
the .gamma.-Carboxyglutamic Acid Domain"; Higashi et al., J. Biochemistry
(1990) 108:654-662.
Waldman, Science 252:1657-1662 (1991).
|
Primary Examiner: Parr; Margaret
Assistant Examiner: Loring; Susan A.
Attorney, Agent or Firm: Lyon & Lyon
Parent Case Text
RELATED APPLICATIONS
This application is a continuation-in-part of Soule and Brunck, entitled
"Blood Coagulation Protein Antagonists And Uses Therefor", filed Oct. 22,
1990, U.S. Ser. No 07/601,454, now abandoned and hereby incorporated by
reference herein, including the drawings attached thereto.
Claims
We claim:
1. Monoclonal antibody 12D10 secreted by a cell line as deposited by ATCC
Accession No. HB 10558.
2. Functional antigen binding fragments of monoclonal antibody 12D10
secreted by a hybridoma cell line as deposited by ATCC Accession No. HB
10558.
3. Hybridoma cell line ATCC HB 10558.
Description
FIELD OF THE INVENTION
The present invention relates to antibodies and functional fragments
thereof and, more particularly, to the treatment of patients for
thrombotic disease or the prevention of thrombotic disease using
anti-thrombotic agents including immunoglobulin protein and protein
fragments or derivatives directed to certain blood coagulation-related
proteinaceous antigens and epitopic regions thereof.
BACKGROUND OF THE INVENTION
Hemostasis is a naturally occurring process which results in the
spontaneous arrest of bleeding from damaged blood vessels. For example,
precapillary vessels will contract immediately when an individual is cut.
Within seconds after such a cut, the process of hemostasis begins. At a
site of injury with disruption of a blood vessel or exposure of
subendothelial vascular tissue, two events rapidly occur. The two limbs of
the hemostatic system, each comprised of many molecules, are activated.
The coagulation (clotting) system is immediately initiated producing
thrombin; and blood platelets adhere to matrix proteins. The platelets are
activated, in part by thrombin, and release adenosine diphosphate ("ADP")
leading to aggregation of additional platelets into a growing platelet
plug in concert with the conversion of fibrinogen in the blood to the
insoluble fibrin gel. This hemostatic plug is strengthened by additional
enzymatic cross-linking. Over time it is dissolved during tissue repair to
result in normal tissue and blood vessel, with or without residual
pathology of the local vessel wall or tissue.
Thrombogenesis is an altered, pathogenic state of one or both limbs of the
hemostatic system. In such states, an intravascular (arterial or venous)
thrombus results from a pathological disturbance of hemostasis. A
platelet-rich thrombus, for example, is thought to be initiated by the
adhesion of circulating platelets to the wall of an arterial vessel. This
initial adhesion, activation by thrombin or other agonists, and the
concomitant release of ADP from platelets, is followed by
platelet-platelet interaction or aggregation. Fibrin formation is
associated with the platelet thrombus but is a minor component. The
arterial thrombus can grow to occlusive proportions in areas of slower
blood flow.
In contrast, fibrin-predominant thrombi develop initially in areas of
stasis or slow blood flow in blood vessels and may resemble a blood clot
formed in vitro. The bulk of venous thrombi comprise a fibrin network
enmeshed with red blood cells and platelets. A venous thrombus
can-establish a "tail" that can detach and result in embolization of the
pulmonary arteries. Thus, it will be understood that arterial thrombi
cause serious disease by local ischemia, whereas venous thrombi do so
primarily by distant embolization.
A platelet plug formed solely by ADP-stimulating platelet interaction is
unstable. Immediately after the initial aggregation and viscous
metamorphosis of platelets, as noted above, fibrin becomes a constituent
of a platelet-rich thrombus. Production of thrombin occurs by activation
of the reactions of blood coagulation at the site of the platelet mass.
This thrombin may activate the initial adherent platelets and stimulates
further platelet aggregations. Platelet aggregation is stimulated not only
by inducing the release of ADP from the platelets, but also by stimulating
the synthesis of prostaglandins, which as aggregating agents are more
powerful than ADP, and by the assembly of the prothrombinase complex on
the activated platelets to accelerate the formation of more thrombin, the
very powerful activator of platelets.
The coagulation of blood results in the formation of fibrin. It involves
the interaction of more than a dozen proteins in a cascading series of
proteolytic reactions. At each step a clotting factor zymogen undergoes
limited proteolysis and itself becomes an active protease. This
clotting-factor enzyme activates the next clotting factor zymogen until
thrombin is formed which connects fibrinogen to the insoluble fibrin clot.
The blood clotting factors include factor I (fibrinogen), factor II
(prothrombin), tissue factor (formerly known as factor III), factor IV
(Ca.sup.2+), factor V (labile factors), factor VII (proconvertin), factor
VIII (antihemophilic globulin, or "AHG"), factor IX (Christmas factor),
factor X (Stuart factor), factor XI (plasma thromboplastin antecedent, or
"PTA"), factor XII (Hageman factor), factor XIII (fibrin-stabilizing
factor), and factors HMW-K (high-molecular-weight kininogen, or Fitzgerald
factor), PRE-K (prekallikrein, or Fletcher factor), Ka (kallikrein), and
PL (phospholipid).
Fibrinogen is a substrate for the enzyme thrombin (factor IIa), a protease
that is formed during the coagulation process by the activation of a
circulating zymogen, prothrombin (factor II). Prothrombin is converted to
the active enzyme thrombin by activated factor X in the presence of
activated factor V, Ca.sup.2+, and phospholipid.
Two separate pathways, called the "intrinsic" and "extrinsic" systems, lead
to the formation of activated factor X. In the intrinsic system, all the
protein factors necessary for coagulation are present in the circulating
blood. In the extrinsic system, tissue factor, which is not present in the
circulating blood, is expressed on damaged endothelium, on activated
monocytes by cells in the arteriosclerotic plaque or by cells outside the
vessel wall. Tissue factor then acts as the receptor and essential
cofactor for the binding of factor VII resulting in a bimolecular enzyme
to initiate the extrinsic pathway of coagulation. This mechanism also
activates the intrinsic pathway of coagulation. The tissue factor pathway
can very rapidly clot blood.
Blood can also be clotted by the contact system via the intrinsic pathway
of coagulation. The mechanism is somewhat slower than the tissue factor
pathways, presumably because of the larger number of reactions that are
required. Both the intrinsic system and extrinsic system pathways must be
intact for adequate hemostasis. See Zwaal, R. F. A., and Hemker, H. C.
"Blood cell membranes and hemostasis." Haemostasis, 11:12-39 (1982).
Thrombosis and a variety of related forms of diseases are associated with,
and result from, activation of one or more of the coagulation protease
cascades pathways, and disorders of regulation of the combined
coagulation/anticoagulation/fibrinolytic pathways. These diseases affect
approximately 2.5 million individuals annually in the United States. Some
three percent of the U.S. population over the age of 45 develop some form
of thrombotic disease or disseminated coagulation each year. Other
thrombotic diseases are hereditary and may affect 100,000 people annually.
Seventy percent of such diseases are fatal by 45 years of age.
Of acquired thrombotic diseases, coronary thrombosis at about 1.5 million
cases per year, pulmonary thromboembolism at about 400,000 cases per year
and severe septic shock at more than 300,000 cases per year, disseminated
intravascular coagulation (DIC) at about 350,000 cases per year, and deep
vein thrombosis at about 175,000 cases per year, predominate. However,
diseases such as menigococemia, hemorrhagic fever virus infections, and a
variety of other diseases produce significant morbidity and mortality as
well. See, e.g., Kaplan, K. "Coagulation Proteins in Thrombosis." In
Hemostasis and Thrombosis, Colman, R. W., et al. eds., pages 1098 et seq.
(2d Ed. J. B. Lippincott Co. 1987). Some of the most acutely severe forms
of disseminated intravascular coagulation affect children secondary to a
variety of infectious diseases. Current treatment for thromboembolic
disease is by no means satisfactory, and includes the use of
anticoagulants, antithrombotic drugs and thrombolytic agents.
One of the most well-known anticoagulants is heparin. Discovered in 1922,
heparin is a heterogenous group of straight-chain anionic
mucopolysaccharides, called glycosaminoglycans, of molecular weights that
average 15,000 daltons. Commercial heparin typically consists of polymers
of two repeating disaccharide units: D-glucosamine-L-iduronic acid and
D-glucosamine-D-glucuronic acid. It is typically prepared from both bovine
lung and porcine intestinal mucosa, and has also been obtained from sheep
and whales.
While heparin occurs intracellularly in mammalian tissues that contain mast
cells, it is limited to a macromolecular form of at least 750,000 daltons.
Furthermore, this heparin has only 10-20% of the anticoagulant activity of
commercial heparin. Heparan sulfate, a compound similar to heparin but
with less anticoagulant activity is a ubiquitous component at the
mammalian cell surface. When native heparin is released from its bound and
inactive state in the metachromatic granules of mast cells, it is ingested
and rapidly destroyed by macrophages. Heparin cannot be detected in the
circulating blood.
When injected intravenously, commercially prepared heparin impairs blood
coagulation. It acts by complexing with antithrombin III, a serine
protease inhibitor that neutralizes several activated clotting factors,
i.e., factors XIIa, kallikrein (activated Fletcher factor), XIa, IX, Xa
and thrombin (IIa). However, it is most active in inhibiting free thrombin
and activated factor X (Xa). Although antithrombin III was thought to be
the only macromolecule able to inactivate thrombin, other plasma proteins
are now known to possess this activity. Antithrombin III can form
irreversible complexes with serine proteases, and, as a result, the above
protein factors are inactivated. Griffith, M. J. "Heparin-Catalyzed
Inhibitors/Protease Reactions: Kinetic Evidence for a Common Mechanism of
Action of Heparin," Proc. Natl Acad Sci USA, 80:5460-5464 (1983). Heparin
markedly accelerates the velocity, although not the extent of this
reaction. A ternary complex is apparently formed between heparin,
antithrombin III, and the clotting factors. Bjork, I., and Lindahl, U.
"Mechanism of the Anticoagulant Action of Heparin" Mol. Cell. Biochem.,
48:161-182 (1982). Low concentrations of heparin increase the activity of
antithrombin III, particularly against factor Xa and thrombin and this
forms the basis for the administration of low doses of heparin as a
therapeutic regimen.
While purified commercial preparations of heparin are relatively non-toxic,
a chief complication of therapy with heparin is hemorrhage. Heparin also
causes transient mild thrombocytopenia in about 25% of the patients,
severe thrombocytopenia in a few, and occasional arterial thrombi. The
mild reactions result from heparin-induced platelet aggregation, while
severe thrombocytopenia follows the formation of hepar-independent
antiplatelet antibodies complexes. It is to be understood that, in all
patients given heparin, platelet counts must be monitored frequently, any
new thrombi might be the result of the heparin therapy, thrombocytopenia
sufficient to cause hemorrhage should be considered to be heparin-induced,
and that thrombosis thought to result from heparin should be treated by
discontinuation and substitution of an agent that inhibits platelet
aggregation and/or an oral anticoagulant.
Severe thrombocytopenia, hemorrhage, and death have occurred even in
patients receiving "low-dose" heparin therapy. Heparin therapy is,
furthermore, contraindicated in patients who consume large amounts of
ethanol, who are sensitive to the drug, who are actively bleeding, or who
have hemophilia, purpura, thrombocytopenia, intracranial hemorrhage,
bacterial endocarditis, active tuberculosis, increased capillary
permeability, all sorts of lesions of the gastrointestinal tract, severe
hypertension, threatened abortion, or visceral carcinoma. Furthermore,
heparin is to be withheld during and after surgery of the brain, eye, or
spinal cord, and is not to be administered to patients undergoing lumbar
puncture or regional anesthetic block. Goodman and Gillman's The
Pharmacological Basis of Therapeutics, pages 1339-1344 (7th ed. 1985).
There are a number of oral anticoagulants that are also available for
clinical use. Many anticoagulant drugs have been synthesized as
derivatives of 4-hydroxycoumarin or of the related compound,
idan-1,3-dione. The essential chemical characteristics of the coumarin
derivatives for anticoagulant activity are an intact 4-hydroxycoumarin
residue with a carbon constituent at the 3 position. There are a number of
differences in the pharmacokinetic properties and toxicities of these
agents, however, and racemic warfarin sodium is the most widely used oral
anticoagulant in the United States.
The major pharmacological effect of oral anticoagulants is inhibition of
blood clotting by interference with the hepatic post translational
modification of the vitamin K-dependent proteins among which are the
clotting factors, i.e., Factors II, VII, IX and X. These drugs are often
called indirect anticoagulants because they act only in vivo, whereas
heparin is termed a direct anticoagulant because it acts in vitro as well.
Again, hemorrhage is the main unwanted effect caused by therapy with oral
anticoagulants, and such therapy must always be monitored. In reported
order of decreasing frequency, complications include ecchymoses,
hematuria, uterine bleeding, melena or hematochezia, epistaxis, hematoma,
gingival bleeding, hemoptysis, and hematemesis. All of the
contraindications described above in regard to the use of heparin apply to
the anticoagulants as well.
Anti-platelet drugs suppress platelet function and are used primarily for
arterial thrombotic disease, whereas anticoagulant drugs, such as warfarin
and heparin suppress the synthesis or function of clotting factors and are
used to control venous thromboembolic disorders. There are a number of
anti-platelet drugs, the most well-known being aspirin. The efficacy of
these agents for acute treatment has, however, not been established and
there is a real problem with aspirin hemorage.
Thrombolytic drugs include streptokinase, urokinase, tissue plasminogen
activator, and APSAC (acylated plasminogen streptokinase complex). These
are proteins which have demonstrated efficacy for the treatment of acute
thrombotic disease. They promote the dissolution of thrombi by stimulating
the conversion of endogenous plasminogen to plasmin, a proteolytic enzyme
that hydrolyzes fibrin. The use of these agents is limited, however, to
acute thrombotic disease. Fibrinolytic agents are used primarily for the
treatment of patients with established coronary arterial thrombosis.
Effective therapy for a variety of forms of intravascular activation of the
coagulation protease cascades, whether thrombosis or the more catastrophic
forms such as those associated with vasomotor collapse (septic shock) and
other forms of disseminated intravascular coagulations are not entirely
satisfactory, and in the case of septic shock is entirely unsatisfactory.
The need for effective therapy that is capable of rapidly arresting
arterial thrombogenesis is recognized as an important therapeutic
deficiency. This is evident from the recent evidence that heparin is
entirely ineffective in preventing rethrombosis of the 11-20% of patients
that rethrombose at the completion of thrombolytic therapy with tissue
plasminogen activator.
The present invention was made in response to these needs and relates to
antagonists of factor VII and specific antagonists of the procoagulant
activity of factor VIIa and the tissue factor:factor VIIa complex. The
invention includes monoclonal-type antibodies produced by cell systems
including bacteria, such as E. coli, or by hybrid cell lines,
characterized in that the antibodies, or functional fragments thereof,
have predetermined specificity to factor VII, to factor VIIa, and/or to
the bimolecular complex of tissue factor and factor VIIa, are effective
for neutralization of these targets, and find application as
antithrombotic agents for syndromes such as disseminated intravascular
coagulation ("DIC") and venous thrombosis. The present invention also
relates to the use of these monoclonal-type antibodies in methods for the
purification of factor VII, factor VIIa and the bimolecular complex
referred to above, and in methods for the immunoassay or immunodetection
of factor VII, factor VIIa and the tissue factor/factor VIIa bimolecular
complex. The purification of factor VII, factor VIIa and the tissue
factor/factor VIIa complex from a biological sample containing these
antigens can be carried out by immuno-affinity chromatography in which the
biological sample is passed through an immunoadsorbant column or slurry
comprising the novel monoclonal-type antibodies or antibody fragments of
this invention bound to a solid base support to thereby selectively adsorb
said antigenic targets. The immunoassay of factor VII, factor VIIa and the
tissue factor/factor VIIa bimolecular complex for determining the presence
or concentration of these target antigens in a biological sample
containing them can be carried out by contacting said sample with a known
amount of the novel monoclonal-type antibody of this invention and
measuring the resulting adsorbed monoclonal antibody.
Factor VII is a vitamin K-dependent zymogen of the active serine protease
VIIa. Factor VII functions to form a complex with tissue factor in blood,
and on conversion to VIIa forms the complex which then activates factor X
by converting factor X to factor Xa. Procoagulant activity is only
associated with the tissue factor:VIIa complex. Free factor VII and free
factor VIIa, as well as the tissue factor:factor VII complex, do not
possess procoagulant activity. Factor VII is a single polypeptide chain of
about 50,000 daltons that can, in a purified system, be activated by
proteolytic cleavage of disulfide bonds by factor Xa, factor IXa, thrombin
and factor XIIa. Takase, T. et al., "Monoclonal Antibodies to Human Factor
VII: A One Step Immunoradiometric Assay for VIIag, J. Clin. Pathol.,
41:337-341 (1988). Human factor VII, when partially or completely
activated, yields a protein comprised of two polypeptide chains linked by
disulfide bridges. Factor VII and VIIa may be used interchangeably in this
document and will be designated VII/VIIa when target interchangeability is
to be indicated.
With the advent of hybridoma technology first developed by Kohler and
Milstein, it is now possible to attempt to generate monoclonal antibodies
which are essentially homogenous compositions having uniform affinity for
a particular binding site. The production of mouse hybridomas by these
investigators is described in Nature, 256:495-497 (1975) and Eur. J.
Immunol., 6:511-519 (1976). Further procedures are described in Harlow,
E., and Lane D., "Antibodies: A Laboratory Manual" (Cold Spring Harbor
Laboratory 1988). According to the hybridoma method, tissue-culture
adapted mouse myelomas cells are fused to spleen cells from immunized mice
to obtain the hybrid cells, called "hybridomas," that produce large
amounts of a single antibody molecule. Generally, animals are injected
with an antigen preparation, and if an appropriate humoral response has
appeared in the immunized animal, an appropriate screening procedure is
developed. The sera from test bleeds of the immunized animal are used to
develop and validate the screening procedure, and after an effective
screen has been established, the actual production of hybridomas is begun.
Several days prior to the fusion, which is generally carried out in the
presence of polyethylene glycol as described by Galfe et al. Nature,
266:550-552 (1977), followed by selection in HAT medium (hypoxanthine,
aminopterin and thymidine) as described by Littlefield, Science,
145:709-710 (1964), animals are boosted with a sample of the antigen
preparation. For the fusion, antibody secreting cells are prepared from
the immunized animal, mixed with the myeloma cells, and fused. After the
fusion, cells are diluted in selective medium and plated in multi-welled
culture dishes. Hybridomas may be ready to test as soon as about one week
after the fusion, but this is not certain. Cells from positive wells are
grown, subcloned, and then single-cells are cloned.
It is understood that hybridoma production seldom takes less than two
months from start to finish, and can take well over a year. The production
of monoclonal antibodies has been described in three stages: (1)
immunizing animals (2) developing the screening procedure and (3)
producing hybridomas. It is also understood that any one of these stages
might proceed very quickly but that all have inherent problems. For
example, while immunization can be carried out with virtually any foreign
antigen of interest, many difficulties arise and variations may be
required for any specific case in order to generate the desired monoclonal
antibodies. Prior to attempting to prepare a given hybridoma, there is no
assurance that the desired hybridoma will be obtained, that it will
produce antibody if obtained, or that the antibody so produced will have
the desired specificity or characteristics. Harlow, E., and Lane, D.,
supra at Chapter 6.
The production of monoclonal antibodies to human factor VII has been
reported, and these reagents are said to have been used to make
immunodepleted plasma or to detect factor VII cross reactive material in
factor VII deficient patients. Id. The production of monoclonal antibodies
to factor VII for their use in a one step, immunoradiometric assay for
factor VII:ag has also been reported. Id. The authors reported the
preparation of three mouse monoclonal antibodies, two of which were said
to bind, to factor VII:ag, and two of which were said to be inhibitors of
factor VII in vitro. See also Howard et al., J. Clin. Chem., 35:1161
(1989). No monoclonal antibodies against either factor VII or factor VIIa
have been described which therapeutically interfere with the binding of
factor VIIa to tissue factor or which neutralize the activity of the
tissue factor/factor VIIa complex.
BRIEF SUMMARY OF THE INVENTION
In accordance with the present invention novel monoclonal-type antibodies
or antibody fragments are provided which can be produced by recombinant
cell lines or by hybrid cell lines, the antibodies being characterized in
that they have certain predetermined specificity to particular targets,
i.e., factor VII, factor VIIa, the bimolecular complex of tissue factor
and factor VIIa, and to particular epitopic regions thereof, and have
neutralizing capability when combined with these targets. By virtue of
their binding to factors VII and VIIa as competitive, non-functional
surrogates of tissue factor, they serve as antagonists to neutralize the
functional activation of the coagulation protease cascades. These
antibodies are useful in the prevention and therapeutic treatment of
thrombotic conditions and related diseases in which the activation of the
above coagulation protease cascades plays a significant pathogenic role.
Particular antibodies are also useful in methods for the purification of
factors VII and VIIa and the tissue factor/factor VIIa bimolecular
complex, and in the immunoassay of these target antigens.
Accordingly, the present invention also provides a method of preventing or
treating a mammalian species for an incipient or existing thrombotic
disease condition that would be alleviated by an agent that selectively
interferes with the extrinsic coagulation cascade, which comprises
administering to a mammalian species in need of such treatment a
prophylactically or therapeutically effective amount of a tissue
factor:factor VIIa complex antagonist. The present invention provides for
the prevention or treatment of thrombotic disease conditions including
acute disseminated intravascular coagulation, septic shock, coronary
thrombosis, organ transplant rejection, and deep vein thrombosis.
Effective tissue factor:factor VIIa complex antagonists include
monoclonal-type antibodies, preferably monoclonal antibodies or fragments
thereof, having the tissue factor:factor VIIa complex antagonist
characteristics of antibodies produced by hybridoma cell line ATCC HB
10558. The invention further provides for monoclonal antibodies having the
ability to complex with all or some portion of a loop region on the factor
VII/VIIa molecule, preferably the structural loop region which comprises
the amino acids 195-208 on the factor VII/VIIa molecule.
The invention also provides for compositions useful in the prevention or
treatment of a thrombotic disease condition which comprises an effective
amount of a tissue factor:factor VIIa complex antagonist. Such
compositions may include the monoclonal antibodies and/or monoclonal
antibody fragments referenced above. The invention further provides for
substantially purified and purified preparations of monoclonal antibodies
or monoclonal antibody fragments which substantially inhibit the
procoagulant activity of the tissue factor:factor VIIa complex. The
present invention also provides for hybridoma cell lines which permit the
production of such monoclonal antibodies and monoclonal antibody
fragments. Methods for producing such hybridoma cell lines are also
described and claimed herein that comprise immunizing an animal species
with an immunogen comprising one or more factor VIIa structural loop
region peptides.
Methods for inhibiting the procoagulant activity of the tissue
factor:factor VIIa complex in vivo are also described and claimed, which
comprise administering to a mammalian species a monoclonal-type antibody
or antibody fragment that specifically reacts with said complex but does
not substantially inhibit free factor VIIa.
The factor VIIa and the tissue factor/factor VIIa bimolecular complex
against which the monoclonal-type antibodies of this invention have
specificity can be isolated from biological samples in the methods
described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
While the specification concludes with claims particularly pointing out and
distinctly claiming the subject matter regarded as forming the present
invention, it is believed that the invention will be better understood
from the following description taken in connection with the accompanying
drawings in which:
FIG. 1 is a schematic representation of the clotting cascade, divided into
those sequences involved in surface (contact) activation, intrinsic and
extrinsic activation, and the final common pathway. Solid lines indicate
direct activation of precursor zymogen to enzyme; interrupted lines show
paths of both positive and negative feedback. PL indicates phospholipid.
FIG. 2 shows the ability of 12D10 monoclonal antibody and 12D10 F(ab)
fragment to inhibit the clotting of recalcified human plasma in a two
stage prothrombin time test.
FIG. 3 is a graph showing plasma PT (prothrombin time) and factor VII
coagulant activity over time following intravenous administration of 12D10
F(ab) fragment.
FIG. 4 is a graph showing PT and factor VII coagulant activity over time
following intravenous administration of 12D10 F(ab) fragment.
FIG. 5 is a graph showing PT and free factor VII/VIIa antigen over time
following intravenous administration of 12D10 F(ab) fragment.
FIG. 6 is a graph showing PT and 12D10 Fab concentration over time
following intravenous administration of 12D10 F(ab) fragment.
FIG. 7 is a graph showing PT before and after administration of 12D10 F(ab)
in a single chimpanzee.
DETAILED DESCRIPTION OF THE INVENTION
As specifically shown in FIG. 1, blood coagulation can begin when the
Hageman factor (XII) undergoes contact activation and becomes bound to
surfaces. This surface-bound factor XII undergoes proteolytic activation
by kallikrein (Ka) in the presence of a high-molecular-weight kininogen
(HMW-K). This surface activation (contact system, intrinsic pathway)
appears to initiate coagulation in vitro but is not considered to be a
relevant in vivo mechanism. Deficiencies in this pathway (XII,
prekallikrein and HMWK) result in prolonged in vitro clotting times but do
not cause hemostatic disorders.
Factor XIIa constitutes an arm of a feedback loop and activates more Ka
from prekallikrein (Pre-K or Fletcher factor), in the presence of HMW-K.
Factor XIIa in the presence of HMW-K also activates factor XI. Factor XIa
in the presence of Ca.sup.2+ proteolytically activates factor IX to IXa.
Factor VIII, factor IXa, Ca.sup.2+, and phospholipid micelles (PL) from
blood platelets form a lipoprotein complex with factor X and result in
factor X activation. Factor V, factor Xa, Ca.sup.2+, and PL also form a
lipoprotein complex with factor II or prothrombin and activate it to IIa
or thrombin. In seconds, thrombin splits two small pairs of peptides off
the large fibrinogen molecule, followed by rapid noncovalent aggregation
of soluble fibrin monomers. Factor XIII, activated by thrombin to XIIIa,
cross-links adjacent fibrin monomers covalently to form the insoluble
fibrin clot.
There is considerable evidence that tissue factor initiates coagulation in
the generalized Schwartzman reaction (DIC) resulting from endotoxinemia.
Kaplan, K. Coagulation Proteins in Thrombosis. In "Hemostasis and
Thrombosis" (Colman, R. W., Hirsh, J., Marder, V. J., and Salzman, E. W.;
eds.) 2nd Ed. J. B. Lippincott Co., pp. 1098, 1987. Fibrin microthrombi
are uniformly found in fatal DIC and thrombosis of large arteries and
veins may be found in 40% of cases. Minna, J. D., Robboy, S. J., Colman,
R. W. Disseminated Intravascular coagulation in Man. C. C. Thomas, 1974.
Leukocytes are required participants and are induced by endotoxin to a
procoagulant (thrombogenic) state, Semararo, N , et al , . "Role of
leukocyte procoagulant activity in endotoxin-induced DIC: Evidence from
comparative studies in rats and rabbits." Agents Actions 11:646, 1981, 26,
expressing tissue factor. Colucci, M., "Cultured human endothelial cells
generate tissue factor in response to endotoxin." J. Clin. Invest.
73:1893, 1983. At the same time endothelial cells are also induced to
express tissue factor, initiate coagulation and to depress their
anticoagulant properties. Moore, K. L., "Endotoxin enhances tissue factor
and suppresses thrombomodulin expression of human vascular endothelium in
vitro." J. Clin. Invest. 79:124-130, 1987.
The extrinsic coagulation cascade as currently envisioned starts with the
formation of the tissue factor:VII and tissue factor:VIIa complexes on the
surface of tissue factor expressing cells. Tissue factor is not normally
expressed by blood cells or vascular endothelial cells, but following
stimulation with LPS, TNFalpha or IL-1, endothelial cells transcribe and
express this molecule. Though fewer molecules of tissue factor are
expressed, factor VII is bound and rapidly converted to factor VIIa by
factor Xa feedback activation of the bound factor VII. Endothelial cell
factor IX/IXa receptor (IX-R) and factor VIII (activated to factor VIIIa
by Xa or thrombin feedback) markedly enhance the kinetics of factor Xa
generation by limited proteolytic activation of factor X. Cell surface
associated factor V (activated by thrombin feedback) further amplifies the
Vmax of factor Xa and prevents inhibition by plasma heparin:AT-III
inhibitor. Prothrombin is efficiently converted to thrombin to convert
fibrinogen to fibrin, leads to release of Plasminogen activator inhibitor
I, serves as a chemotactic agent, aggregates platelets, activates Mac-1
receptor of monocytes, and has other inflammatory effects.
There are no presently effective drugs for the inhibition of the extrinsic
pathway. The use of heparin, shown to be without benefit, is nevertheless
continued clinically with the attendant secondary problems of its platelet
effects. In DIC with depletion of antithrombin III there is no benefit to
heparin since it is not a direct anticoagulant, only a cofactor for the
thrombin inhibitor antithrombin III when present as a
[heparin:antithrombin-III] complex. Anti-platelet drugs do not inhibit the
coagulation protease cascade, and they diminish the necessary hemostatic
properties of platelets. Warfarin therapy to interfere with Vitamin K
supported gamma carboxylation of factors VII, X, IX and prothrombin is too
slow and is associated with reduced activity of the natural
anticoagulation pathways due to inhibition of gamma carboxylation of
Protein C and Protein S. The present invention addresses this need by
inhibiting the reaction pathway at the earliest possible step, the
initiating proteolytic complex of tissue factor:VIIa, which will block the
intravascular initiation of coagulation by tissue factor positive cells,
e.g., endothelial cells, monocytes, and tissue factor positive foam cells
in atherosclerotic plaques.
The present invention employs a neutralizing antagonist surrogate cofactor,
preferably a monoclonal antibody or antibody fragment, to the functional
bimolecular initiation complex of tissue factor and VII/VIIa and, more
preferably, to a neoantigen(s) induced on the bimolecular tissue
factor:VIIa complex or alternatively, a neutralizing monoclonal antibody
to VII or VIIa, preferably to a structural loop region thereof. Binding of
such monoclonal antibodies to tissue factor:VIIa so as to block the active
site of VII/VIIa, dissociate VIIa from tissue factor or competitively
inhibit the association of the substrate serine protease zymogens factors
X or IX will inhibit initiation of coagulation on vascular cells and
arrest one of the major pathogenetic processes in thrombotic diseases.
Though the activation of coagulation has long been recognized as central
and required for thrombus formation and growth and for disseminated
intravascular coagulation, many mechanisms are put into action,
particularly in the most virulent forms of septic shock. It has been
demonstrated that monoclonal antibodies to TNFalpha are capable of
protecting baboons from endotoxin mediated septic shock, Tracey K. J.,
"Anti-cachectin/TNF monoclonal antibodies prevent septic shock during
lethal bacteraemia." Nature 330:662, 1987, since TNFalpha is induced by
endotoxin, IL-1 and the toxic shock toxin 1. Michie, H. R., "Detection of
circulating tumor necrosis factor after endotoxin administration." N. Eng.
J. Med. 318:1481, 1988, Jupin, C., et al., "Protein C prevents the
coagulopathic and lethal effects of escherichia coli infusion in the
baboon." J. Clin. Invest. 79:18, 1987. However, anti-TNFalpha or anti-LPS
monoclonal antibodies may have low efficacy once the pathologic process
has been established. Recently, it has been shown that activated protein
C, the natural anticoagulant protein given in massive doses is capable of
arresting and even reversing early ongoing septic shock. Taylor, F. B.,
"Protein C prevents the coagulopathic and lethal effects of escherichia
coli infusion in the baboon." J. Clin. Invest. 79:918, 1987. Now, evidence
from the same group with the same model indicates that arresting the
initiation of coagulation with monoclonal antibody to tissue factor is
effective in treating septic shock in lethal challenge of baboons with E.
coli (Edgington, et al., "Tissue Factor: Molecular Biology and
Significance in the Pathophysiology of Gram-Negative Septic Shock," In:
Microbiological, Chemotherapeutical and Immunological Problems in High
Risk Patients. E. Garaci, et al., Eds., Raven Press, New York, Vol. 61,
pp. 29-37 (1989).
One method useful for the production of anti-protein antibodies involves
the use of synthetic peptides from regions of the protein sequence occur
on the surface of the protein to raise and/or screen for desirable
antibodies. In the case of Factor VIIa however, there is no available
experimental data on the structure. Only the amino acid sequence is known.
Factor VIIa has some sequence and structural homology in its catalytic
domain to several other proteases whose structures have been determined by
X-ray crystallography. The sequences of these proteases were analyzed and
the sequence of the catalytic domain of factor VIIa was compared. Regions
of the factor VIIa molecule were discovered that were highly conserved in
structure often representing the core structure of the protein, as well as
regions that were more variable. The regions in sequence with variable
structures, herein denominated "loops," were discovered on the surface of
the catalytic domains.
Eleven loop regions were identified in the sequence of the catalytic domain
of factor VIIa. The include peptides comprising amino acids 165-177,
195-208, 209-218, 234-248, 248-258, 263-278, 285-295, 313-321, 330-339,
348-360, and 367-390. A computer model of the structure of the catalytic
domain of factor VIIa was constructed and the location of the structurally
variable loops ascertained. One set of loops was discovered to be located
near the catalytic site of factor VIIa, and another clustered around the
activation site where various proteases cleave the enzymatically inactive
single-chain form, factor VII, to the active two-chain form, factor VIIa.
Anti-factor VII/VIIa antibody epitopes targeted for neutralization, were
used to generate antibodies subsequently determined to neutralize or
inhibit the activity of Factor VIIa by binding to a loop region. When
these loops are near the active site, binding of these antibodies blocks
access, for example, to the site by substrates such as Factor X and
thereby inhibits the function of factor VIIa. In this manner, antibodies
are prepared that block the extrinsic coagulation pathway.
Description of hybridoma preparation and initial characterization of
monoclonal antibodies against factor VII/VIIa and the tissue factor/factor
VIIa complex, is set forth in Example 1 below. Parameters are described
relating to preparation of the antigen, dose and form of antigen, route of
inoculation and immunization protocol, hybridoma preparation, and the
screening, isolation and initial characterization of monoclonal
antibodies. The properties of a monoclonal antibody designated 12D10 (ATCC
HB 10558) are set forth and described. The antibody was shown to bind
factor VII/VIIa and to dramatically inhibit the activity of the tissue
factor:factor VIIa complex. As shown by the results in Example 2, the
12D10 antibody was also able to inhibit the activity of free factor VIIa.
The 12D10 monoclonal antibody was shown in Example 3 to be specific to
amino acids 195-208 region of the factor VII/VIIa molecule. Fragmentation
of the 12D10 monoclonal antibody as described in Example 5, furthermore,
was beneficially shown not to affect its clotting inhibition activity.
Antibodies, or the desired binding portions thereof including F(ab) and Fv
fragments, can also be generated using processes which involve cloning an
immunoglobulin gene library in vivo. Huse et al , . "Generation of a Large
Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda,"
Science 246:1275-1281 (Dec. 8, 1989). Using these methods, a vector system
is constructed following PCR amplification of messenger RNA (mRNA)
isolated from spleen cells with oligonucleotides that incorporate
restriction sites into the ends of the amplified product. Separate heavy
chain and light chain libraries are constructed and may be randomly
combined to coexpress these molecules together and screened for antigen
binding. Single chain antibodies may also be prepared and utilized.
Additional monoclonals that neutralize the factor VIIa-tissue factor
bimolecular cell surface activation complex can be made and selected from
three classes of antithrombotic monoclonal antibodies. The specificity of
the three classes of antibody include those reactive with factor VII and
VIIa and neutralize amidolytic activity. Two subsets of antibody are
generated. One will inhibit factor VIIa activity by preventing the
association of tissue factor and factor VII/VIIa and the other will
directly inhibit the activity of factor VIIa. The second class includes
those monoclonals reactive with only Factor VIIa and neutralize amidolytic
activity, while the third includes those reactive with neutralizing
neoepitopes expressed as the result of association of tissue factor and
factor VII. These neoepitopes would not be expressed on either free tissue
factor or factor VII and therefore restricted to the coagulation
initiation complex.
Each of the three classes of antibody represent further unique mechanistic
approaches for antithrombotic therapy. Antibodies in the first class are
defined by the specificity of the 12D10 monoclonal. Antibodies in the
second specificity class are developed by immunizing mice with recombinant
factor VIIa. Monoclonals that react with factor VIIa but not factor VII
are selected. The desirable reagent will inhibit activity of preformed
tissue factor:factor VIIa complexes. Neoepitopes expressed on the
functional bimolecular complex will be immunogenic targets for the
development of monoclonals that neutralize activity. These antibodies are
prepared by performing in vitro immunization of murine splenocytes using
preformed complexes of tissue factor:factor VIIa in an optimal environment
of phospholipids. In vitro immunization is preferred due to the
proteolytically labile nature of the complex in vivo; however, standard in
vivo immunization of heparinized mice can also be employed. Screening is
used to identify only those antibodies reactive with the tissue
factor:factor VIIa complex, as opposed to those that are reactive with
either free tissue factor factor VIIa. These antibodies will only inhibit
coagulation at the site of injury or activation and normal hemostasis will
not be compromised.
EXAMPLE 1
Preparation of hybridomas and identification of desired monoclonal
antibodies was as follows. Female balb/c mice were immunized with purified
human factor VII (factor VII) isolated from pooled human plasma over a
period of approximately six months. Complete Freund's adjuvant was used
for primary immunization and incomplete Freund's adjuvant for booster
immunization. One to ten micrograms of protein was used per immunization.
Route of immunization was both intraperitoneal and subcutaneous. Three
days prior to fusion mice received an intravenous perfusion boost of
purified factor VII (20 .mu.g) in saline. Spleens were removed and spleen
cells were fused to the SP2/0 myeloma following standard hybridoma
methods.
The screening strategy employed a three-staged methodology. Primary
screening identified hybridoma antibodies that reacted with factor VII or
factor VIIa antigen. Secondary screening identified antibodies capable of
inhibiting the functional activity of factor VIIa as indirectly assessed
in a factor X activation chromogenic substrate assay. Tertiary screening
assessed clotting inhibition of recalcified plasma in a two-stage
prothrombin time test.
The primary screening assay was a radioimmunoassay where antibodies were
tested for binding to .sup.125 I-factor VII. Briefly, ninety-six well
polyvinyl chloride microliter plates were passively coated with affinity
purified goat anti-mouse IgG obtained from Sigma Chemical Company, St.
Louis, Mo. Antibodycoated plates were blocked with bovine albumin and
culture supernatants (diluted at least 1:50) were bound to the plates.
Plates were washed to remove unbound antibody and .sup.125 I-factor VII or
factor VIIa (100,000 cpm/well; specific activity of factor VII=6
.mu.i/.mu.g; specific activity factor VIIa=4 .mu.Ci/.mu.g) was added
followed by incubation. Plates were washed to remove unbound factor VII
and wells were transferred to a gamma counter to determine bound labeled
factor VII. Negative controls include hybridoma culture supernatant from a
cell line secreting irrelevant monoclonal antibody such as anti-tPA,
sterile culture medium and buffer. Competition of binding of .sup.125
I-factor VII to antibody with excess unlabeled ligand is used to further
demonstrate specificity.
The second screening was used to evaluate the ability of isolated
antibodies to inhibit tissue factor catalyzed factor VII activity as
reflected by the conversion of factor X to factor Xa. The human bladder
carcinoma cell line J-82 (ATCC HTB-1) expresses cell surface-associated
tissue factor and is used as the source of tissue factor and
phospholipids. A chromogenic substrate for factor Xa is used, which
develops color proportional to the amount of factor VIIa activity.
Conversely, color is not developed if factor VII activity is blocked. The
assay is performed as follows. J-82 cells are suspended in tris-buffered
saline at a concentration of 1.times.10.sup.5 cells per mL. Fifty
microliters of cell suspension is added to individual wells of a 96-well
polystyrene microliter plate. Fifty microliters of hybridoma culture
supernatant diluted at least 1:10 is added to appropriate wells followed
by 25 .mu.L of 20 mM CaCl.sub.2. Negative control is irrelevant hybridoma
culture supernatant (such as anti-tPA supernatant) and positive control is
1 .mu.M PPACK (d-phenylalanine-proline-arginine-chloromethylkefone).
Twenty-five microliters of 90 nM factor X and 50 .lambda.L of substrate
Spectrozyme Xa are added to each well. Following thirty minute incubation
at room temperature, OD-405 is determined. Maximum activation (negative
control) was obtained with samples that were treated with buffer or
irrelevant hybridoma culture supernatant. Complete inhibition (positive
control) assessed with the PPACK from this assay is shown in Table 1
below.
TABLE 1
______________________________________
Results of Factor X activation assay
Sample Treatment OD-405
______________________________________
Buffer 1.101
Anti-tPA hybridoma
1.151
culture supernatant
(1:10)
PPACK (1 .mu.M) 0.023
______________________________________
The properties a monoclonal antibody isolated from a preferred hybridoma,
designated 12D10, in the factor VII/VIIa binding assay and the factor X
activation assay are show in Tables 2 and 3 below. The 12D10 cell line was
deposited with the American Type Culture Collection, 12301 Parklawn Drive,
Rockville, Md. 20852, on Sep. 25, 1990, under the provisions of the
Budapest Treaty.
TABLE 2
______________________________________
Identification of Hybridoma ANtibody 12D10
in F.VII/VIIa Antigen Binding Assay
Antibody .sup.125 I-rec Antigen
CPM Bound
______________________________________
12D10 rec* F.VII 70397
12D10 rec F.VIIa 30489
12D10 rec F.VII + 50-fold molar excess
1878
cold rec F.VII
12D10 rec VIIa + 50-fold molar excess
2771
cold rec F.VIIa
anti-tPA rec F.VII 5419
anti-tPA rec F.VIIa 3734
Culture medium
rec. F.VII 2232
Culture medium
rec VIIa 4311
______________________________________
*rec = recombinant
Results in Table 2 show that the 12D10 monoclonal antibody reacts
specifically with both factor VII and factor VIIa antigen.
TABLE 3
______________________________________
Identification of Neutralizing Activity
of Hybridoma antibody 12D10
Percent Inhibition of F.X
Inhibitor Activation
______________________________________
Buffer 0
Anti-tPA Monoclonal Antibody
3
PPACK (1 .mu.M) 100
12D10 (1:10 dilution)
100
______________________________________
Results in Table 3 indicate that the 12D10 monoclonal antibody inhibits
activity of the tissue factor:factor VIIa complex.
EXAMPLE 2
The factor X activation assay was used to determine the mechanism of
inhibition by the 12D10 monoclonal antibody. Hybridoma culture
supernatants diluted 1:50 were preincubated with either rF.VIIa or rF.VIIa
(rF.7VIIa and rF.VIIa indicates recombinant source of specified molecule)
precomplexed to cellular tissue factor expressed on the surface of J-82
cells. Antibody incubations were for 30 minutes at room temperature.
Efficiency of antibody blocking was assessed in the factor X activation
assay. Controls included irrelevant hybridoma antibody (anti tPA) and a
monoclonal to factor VIIa which is known to prevent the association of
tissue factor with factor VIIa but will not inhibit activity following
complex formation (Mab 1296). The optimal specificity for an
antithrombotic monoclonal antibody is one that inhibits the cellular
complex of tissue factor and factor VIIa. Results from this experiment are
presented in Table 4.
TABLE 4
______________________________________
Mechanism on Inhibition of F.VIIa
by Hybridoma Antibody 12D10
Monoclonal
Antibody Preincubation
% Inhibition of F.X Activation
______________________________________
12D10 F.VIIa 97 .+-. 0
12D10 TF:VIIa 98 .+-. 0
1296 F.VIIa 85 .+-. 1
1296 TF:VIIa 22 .+-. 12
anti tPA F.VIIa 0 .+-. 0
anti tPA TF:F.VIIa 3 .+-. 5
______________________________________
These results demonstrate the ability of monoclonal antibody 12D10 to
inhibit the activity of both free factor VIIa and cellular complexes of
tissue factor and factor VIIa, an important property for the disclosed
therapeutic anticoagulant.
EXAMPLE 3
Detailed specificity of the 12D10 monoclonal antibody was evaluated as
follows. A series of synthetic peptides representing linear sequences from
the two gla domains, EGF domains, the light chain and the catalytic site
of factor VIIa were tested for reactivity with the 12D10 monoclonal
antibody. The experiment was performed by coating microtiter wells with
12D10 monoclonal antibody and reacting the capture antibody with 25 .mu.L
of a 100 .mu.M solution of indicated peptide for 30 minutes at 37.degree.
C. following this incubation, 25 .mu.L of 1 nM.sup.125 I-factor VIIa was
added to each well for one hour at room temperature. Wells were washed and
bound .sup.125 I-factor VIIa was determined. Factor VIIa peptide
containing amino acid residues 195-208 prevented the binding of the 12D10
monoclonal antibody to .sup.125 I-factor VIIa.
This result was confirmed by direct binding of the 12D10 monoclonal
antibody to the Factor VIIa peptide 195-208. Peptides were adsorbed to
microtiter wells at a concentration of 1 mg per mL at 37.degree. C. for 2
hours. Wells were blocked with albumin and reacted with 12D10 monoclonal
antibody (10 .mu.g per mL) for 2 hours at 37.degree. C. Goat anti-mouse
IgG peroxidase conjugate was used to demonstrate bound monoclonal antibody
followed by substrate development and determination of OD-450. Peptides
representing the gla (2 peptides), EGF (8 peptides), light chain (2
peptides) and catalytic (11 peptides) domains were tested. The catalytic
domain peptide 195-208 bound 12D10 monoclonal antibody (OD-450=0.450)
while all others were negative (OD-450.ltoreq.0.042). The specificity of
the 12D10 monoclonal antibody for the catalytic domain of factor VIIa is
consistent with our discovery that the antibody binds to VIIa before and
after reaction with tissue factor:VII complex.
EXAMPLE 4
Characterization of the activity of F(ab) fragments of 12D10 antibody was
carried out as follows. The production of F(ab) fragments of the 12D10
monoclonal antibody was accomplished using a commercial kit (Bioprobe
International Tustin, CA). F(ab) fragments are prepared by papain cleavage
of IgG. Papain is inhibited and removed by addition of anti-papain
polyclonal antibody. Protein A chromatography is used to clear Fc
fragments, intact IgG and immune complexes containing papain. The F(ab)
fragments were further purified by size exclusion chromatography using a
Superose 12 column. Monoclonal antibody 12D10, purified as described
above, was analyzed before and after papain digestion. The resulting
purity of the 12D10 IgG and F(ab) fragments was about 95%. The activity of
these F(ab) fragments was compared to intact 12D10 IgG in both the factor
X activation assay and clotting inhibition assays. Analysis of 12D10 IgG
and F(ab) fragments in the factor X activation assay was performed as
described above, except that optimally relipidated recombinant human
tissue factor was substituted for J-82 cells as a source of tissue factor.
This modification enhances the precision and the reproducibility of the
assay and alleviates the need to perform cell culture to obtain J-82
cells. In these experiments, factor VII and was preincubated for 30
minutes at room temperature with the 12D10 IgG or F(ab) prior to
introducing the immune complexes into the factor X activation or clotting
assays.
TABLE 5
______________________________________
Inhibition of F.X Activation With 12D10 IgG and F(ab)
Molar Ratio of
Antibody Binding
Inhibitor Sites to Factor VIIa
Percent Inhibition
______________________________________
12D10 IgG 100 100
12D10 IgG 10 100
12D10 IgG 1.0 100
12D10 IgG 0.1 28
12D10 F(ab) 100 100
12D10 F(ab) 10 100
12D10 F(ab) 1.0 100
12D10 F(ab) 0.1 45
Anti-tPA IgG
100 5
Anti-tPA IgG
10 0
Anti-tPA IgG
1.0 2
Anti-tPA IgG
0.1 0
______________________________________
These results, shown in Table 5, indicate that the fragmentation of the
12D10 antibody did not result in a loss of biological activity. Activity
of the IgG and F(ab) fragments are essentially identical under these
experimental conditions. The potency of the 12D10 antibody is evident from
this result as inhibition at 1:1 molar ration of antibody site to enzyme
is observed. Inhibition of the factor X activation assay at ratios of
factor VIIa:monoclonal less than 1 can be explained by the fact that the
assay readout is an extreme amplification of the residual (uninhibited)
quantity of factor VIIa activity present in the sample.
EXAMPLE 5
The 12D10 monoclonal antibody will inhibit the clotting of recalcified
human plasma in a two stage prothrombin time test. The effect of the F(ab)
fragmentation process on this property was analyzed as follows. Plasma was
diluted 1:2 with 2 mM sodium citrate. IgG or F(ab) at indicated
concentrations (50 .mu.L) was added to 100 .mu.L of diluted plasma and
incubation was performed at room temperature for 20 minutes. Human
thromboplastin (Thromborel S: Behring Diagnostics) was diluted 1:1000 in
30 mM CaCl.sub.2 and 200 .mu.L was added to the plasma/antibody solution.
Clotting times were determined in a Coagamate optical coagulometer (Organon
Technica). Clotting times are converted to percent tissue factor activity
by an algorithm previously described, Hvatum, M. and Prydz H. Studies on
Tissue Thromboplastin. Solubilization with Sodium Dioxycholate. Biochem.
Biophys. Acta. 130:92-101 (1966). The results are presented in FIG. 2.
These results demonstrate that the 12D10 F(ab) fragment is a potent
inhibitor of clotting of human plasma.
EXAMPLE 6
The 12D10 monoclonal antibody decreases the level of free factor VII/VIIa
antigen upon administration in vivo to a chimpanzee. Such a decreased
level of these antigens is maintained for periods in excess of one hour,
and in some cases for over five, or even ten hours.
The F(ab) fragment of the 12D10 monoclonal antibody was administered to
chimpanzees to assess pharmacokinetics, systemic anticoagulant effect and
limited toxicity. Three normal adolescent chimpanzees (45-55kg) were
selected for the study. All animals underwent physical examination by a
veterinarian including complete blood count (CBC), serum chemistry screen,
fecal exam (oocytes and parasites) and hepatitis serological screens.
Hematology, coagulation and clinical chemistry parameters were tested and
shown to be within normal range. Animals were selected that had no
previous exposure to murine proteins, and that were chimp anti-murine
antibody (CHAMA) negative.
Animals were intermittently anesthetized with ketamine prior to
manipulations. Initially, five doses were selected for administration.
Doses that prolonged the prothrombin time were repeated in two additional
chimpanzees. Plasma samples were obtained at -15, 15, 30, 60, 120, 240,
360, 1440 and 2880 minutes following administration for evaluation of
hematologic, physiologic and coagulation parameters. The study required
five weeks with a total of 16 injections of the 12D10 Fab fragment. No
immediately observable ill effects were noted; however, some hematoma
formation and bleeding back at the site of ketamine injection was observed
at the higher doses.
Administration of low levels (0.0023-0.0058 mg per kg) had no apparent
effect on PT or factor VII activity (both measured using standard assays).
FIG. 3 illustrates that PT values and factor VII coagulant activity remain
essentially constant following injection with a 0.0035 mg per kg dose of
the 12D10 F(ab) fragment.
The threshold for observation of systemic effect was at a dose of 0.04 mg
per kg. At this dose level, the prothrombin time was prolonged to greater
than 30 seconds, with simultaneous reduction of factor VII coagulant
activity and free factor VII/VIIa antigen to less than 10% and less than
10ng per mL, respectively.
The systemic effect of 12D10 F(ab) fragment on coagulation parameters was
observed in a chimpanzee infused intravenously with 0.0547 mg per kg 12D10
F(ab) fragment. In FIG. 4, the rapid increase in plasma PT values was
reflected by the concomitant decrease in factor VII coagulant activity.
The decrease in free factor VII/VIIa antigen level is demonstrated in FIG.
5.
As factor VII/VIIa antigen and factor VII coagulant levels fell, plasma
levels of 12D10 F(ab) fragment increased dramatically in parallel to the
PT values over the course of the study. Plasma F(ab) fragment levels
appeared to correlate directly with plasma PT values and in reverse with
factor VII/VIIa antigen levels. Plasma F(ab) fragment reached peak
concentration within the first 15 minutes following injection. The change
in plasma levels of 12D10 F(ab) fragment is shown in FIG. 6.
FIG. 7 compares a range of doses that were administered to a single
chimpanzee. The duration of response increased with escalating dose level.
The duration of response at the 0.1 mg per kg level was approximately
double that at the 0.0547 mg per g/kg dose level. A dose escalation effect
is also evident from these data. The threshold for this animal appears to
be approximately 0.04 mg per kg 12D10 F(ab) fragment.
Other embodiments are within the following claims.
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